Search PubMed⌕ Search

Biomedical subjects

A C Nairn

Publications and source records attributed to A C Nairn.

At least 19 recordsLinked to original sources
PubMed2006

Cocaine self-administration in mice is inversely related to phosphorylation at Thr34 (protein kinase A site) and Ser130 (kinase CK1 site) of DARPP-32.

The reinforcing effect of cocaine is associated with increases in dopamine in the striatum. The phosphoprotein DARPP-32 (dopamine- and cAMP-regulated phosphoprotein) has been shown to mediate the intracellular events after activation of dopamine receptors. DARPP-32 is phosphorylated at multiple sites by different protein kinases, but little is known about the functional role of these different sites. Cocaine self-administration and striatal levels of dopamine after acute "binge" cocaine administration were measured in separate lines of mice with alanine mutations introduced into DARPP-32 at either Thr34 (protein kinase A site, Thr34A), Thr75, (cyclin-dependent kinase 5 site, Thr75A), Ser97 (kinase CK2 site, Ser97A), or Ser130 (kinase CK1 site, Ser130A). Acquisition of stable cocaine self-administration required significantly more time in Thr34A-/- mice. Both Thr34A- and Ser130A-DARPP-32 mutant mice self-administered more cocaine than their respective wild-type controls. Also, cocaine-induced increases of dopamine in dorsal striatum were attenuated in the Thr34A- and Ser130A-DARPP-32 phosphomutant mice compared with wild-type mice. Notably, levels of P-Thr34- and P-Ser130-DARPP-32 were reduced after self-administration of cocaine in wild-type mice. Thus, phosphorylation states of Thr34- and Ser130-DARPP-32 play important roles in modulating the reinforcing effects of cocaine.

Alanine↗
PubMed2006

2-Deoxyglucose and NMDA inhibit protein synthesis in neurons and regulate phosphorylation of elongation factor-2 by distinct mechanisms.

Cerebral ischaemia is associated with brain damage and inhibition of neuronal protein synthesis. A deficit in neuronal metabolism and altered excitatory amino acid release may both contribute to those phenomena. In the present study, we demonstrate that both NMDA and metabolic impairment by 2-deoxyglucose or inhibitors of mitochondrial respiration inhibit protein synthesis in cortical neurons through the phosphorylation of eukaryotic elongation factor (eEF-2), without any change in phosphorylation of initiation factor eIF-2alpha. eEF-2 kinase may be activated both by Ca(2+)-independent AMP kinase or by an increase in cytosolic Ca2+. Although NMDA decreases ATP levels in neurons, only the effects of 2-deoxyglucose on protein synthesis and phosphorylation of elongation factor eEF-2 were reversed by Na(+) pyruvate. Protein synthesis inhibition by 2-deoxyglucose was not as a result of a secondary release of glutamate from cortical neurons as it was not prevented by the NMDA receptor antagonist 5-methyl-10,11-dihydro-5H-dibenzo-(a,d)-cyclohepten-5,10-imine hydrogen maleate (MK 801), nor to an increase in cytosolic-free Ca2+. Conversely, 2-deoxyglucose likely activates eEF-2 kinase through a process involving phosphorylation by AMP kinase. In conclusion, we provide evidence that protein synthesis can be inhibited by NMDA and metabolic deprivation by two distinct mechanisms involving, respectively, Ca(2+)-dependent and Ca(2+)-independent eEF-2 phosphorylation.

Animals↗
PubMed2005

Control of the CFTR channel's gates.

Unique among ABC (ATP-binding cassette) protein family members, CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, encoded by the gene mutated in cystic fibrosis patients, functions as an ion channel. Opening and closing of its anion-selective pore are linked to ATP binding and hydrolysis at CFTR's two NBDs (nucleotide-binding domains), NBD1 and NBD2. Isolated NBDs of prokaryotic ABC proteins form homodimers upon binding ATP, but separate after hydrolysis of the ATP. By combining mutagenesis with single-channel recording and nucleotide photolabelling on intact CFTR molecules, we relate opening and closing of the channel gates to ATP-mediated events in the NBDs. In particular, we demonstrate that two CFTR residues, predicted to lie on opposite sides of its anticipated NBD1-NBD2 heterodimer interface, are energetically coupled when the channels open but are independent of each other in closed channels. This directly links ATP-driven tight dimerization of CFTR's cytoplasmic NBDs to opening of the ion channel in the transmembrane domains. Evolutionary conservation of the energetically coupled residues in a manner that preserves their ability to form a hydrogen bond argues that this molecular mechanism, involving dynamic restructuring of the NBD dimer interface, is shared by all members of the ABC protein superfamily.

ATP-Binding Cassette Transporters↗
PubMed2005

A molecular switch for translational control in taste memory consolidation.

In a variety of species memory consolidation following different learning paradigms has been shown to be dependent on protein synthesis. However, it is not known whether modulation of protein synthesis is a critical component of the consolidation process, nor is the identity of any protein(s) subject to translational regulation, known. We report here that phosphorylation of eukaryotic elongation factor-2 (eEF2), an indicator for translational elongation attenuation, is correlated with input that produces taste memory consolidation in the relevant cortex of rat. The temporal pattern of eEF2 phosphorylation is similar to extra-cellular regulated kinase 2 (ERK2) activation and S6K1 phosphorylation, which are known to stimulate translation initiation. In addition, increased eEF2 phosphorylation and increased alphaCaMKII expression is detected in a synaptoneurosomal fraction made from taste cortex following memory consolidation. These results suggest that increased initiation rate together with decreased elongation rate, during memory consolidation, shift the rate-limiting step of protein synthesis, to produce a local switch-like effect in the expression of neuronal proteins.

Animals↗
PubMed2004

A network of control mediated by regulator of calcium/calmodulin-dependent signaling.

Calmodulin (CaM) is a major effector for the intracellular actions of Ca2+ in nearly all cell types. We identified a CaM-binding protein, designated regulator of calmodulin signaling (RCS). G protein-coupled receptor (GPCR)-dependent activation of protein kinase A (PKA) led to phosphorylation of RCS at Ser55 and increased its binding to CaM. Phospho-RCS acted as a competitive inhibitor of CaM-dependent enzymes, including protein phosphatase 2B (PP2B, also called calcineurin). Increasing RCS phosphorylation blocked GPCR- and PP2B-mediated suppression of L-type Ca2+ currents in striatal neurons. Conversely, genetic deletion of RCS significantly increased this modulation. Through a molecular mechanism that amplifies GPCR- and PKA-mediated signaling and attenuates GPCR- and PP2B-mediated signaling, RCS synergistically increases the phosphorylation of key proteins whose phosphorylation is regulated by PKA and PP2B.

Animals↗
PubMed2003

Adenylyl cyclase-dependent form of chemical long-term potentiation triggers translational regulation at the elongation step.

The persistent maintenance of long-term potentiation requires both messenger RNA and protein synthesis. While there is mounting evidence for an active role of protein synthesis in hippocampal long-term potentiation, the nature of mechanisms underlying its regulation has not yet been established. We used a previously described chemical long-term potentiation protocol [J Neurosci 19 (1999) 2500] to address the hypothesis that signaling mechanisms, involved in long-lasting long-term potentiation, directly regulate protein synthesis. Chemical long-term potentiation is an N-methyl-D-aspartate receptor-dependent form of plasticity, which relies on both synaptic activity, in the form of spontaneous bursting induced by high concentrations of K(+) and Ca(2+), and cyclic AMP/adenylyl cyclase signaling. We found that chemical long-term potentiation in CA1 of the mouse hippocampus lasts for at least 3 hours and requires both messenger RNA and protein synthesis. However, surprisingly de novo total protein synthesis was paradoxically decreased at 1 hour after long-term potentiation induction. Consistent with the decrease in total protein synthesis in potentiated CA1, phosphorylation of eukaryotic elongation factor 2 was increased and is likely responsible for inhibition of translation at the elongation step. Increased phosphorylation of eukaryotic elongation factor 2 was dependent on coincident cyclic AMP/adenylyl cyclase activation and synaptic activity and required N-methyl-D-aspartate receptor activation. Despite the inhibition in total protein synthesis, the level of the immediate early gene protein Arc (activity regulated cytoskeleton-associated protein) increased at 1 hour after chemical long-term potentiation induction. Taken together, the results suggest that regulation at the elongation step of protein synthesis contributes to persistent forms of long-term potentiation.

Adenylyl Cyclases↗
PubMed2002

N-Methyl-D-aspartate receptor activation inhibits protein synthesis in cortical neurons independently of its ionic permeability properties.

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate, strongly depresses protein synthesis. We have previously demonstrated in cortical neurons that a glutamate-induced increase in intracellular Ca(2+) is likely responsible for the blockade of the elongation step of protein synthesis. In this study, we provide evidence indicating that NMDA mobilizes a thapsigargin-sensitive pool of intracellular Ca(2+). Exposure of cortical neurons to NMDA, in the absence of external Ca(2+), produced a transient rise in intracellular Ca(2+) that was suppressed by pretreatment with thapsigargin. This rise in intracellular Ca(2+) did not result from an influx of Na(+) via reversal of the mitochondrial Na(+)/Ca(2+) exchanger since it persisted in a Na(+)-free medium or in the presence of CGP 37157, an inhibitor of the exchanger. Moreover, the NMDA-induced increase in intracellular Ca(2+) required the presence of D-serine, was blocked by D(-)-2-amino-5-phosphonopentanoic acid, but was not reduced in the presence of external Mg(2+). This unexpected non-ionotropic effect of NMDA was associated with an inhibition of protein synthesis that was also insensitive to the absence of external Ca(2+) or Na(+), or presence of Mg(2+). NMDA treatment resulted in an increase in the phosphorylation of eEF-2 in the absence or presence of external Ca(2+). The initiation step of protein synthesis was not blocked by NMDA since the phosphorylation of initiation factor eIF-2alpha subunit was not altered by NMDA treatment. In conclusion, we provide evidence indicating that NMDA can inhibit protein synthesis in cortical neurons through a process that involves the mobilization of intracellular Ca(2+) stores via a mechanism that is not linked to the ionic properties of NMDA receptors.

Animals↗
PubMed2001

A novel cyclin provides a link between dopamine and RNA processing.

Regulation of gene expression by dopamine may play an important role in learning, reward, and addiction. Hyman and colleagues now report the characterization of ania-6, a novel cyclin that associates with RNA polymerase II and is induced by dopamine or cocaine in the neostriatum. Ania-6 may thus provide a link between dopamine and gene expression at the level of mRNA processing.

Alternative Splicing↗
PubMed2001

Opposing changes in phosphorylation of specific sites in synapsin I during Ca2+-dependent glutamate release in isolated nerve terminals.

Synapsins are major neuronal phosphoproteins involved in regulation of neurotransmitter release. Synapsins are well established targets for multiple protein kinases within the nerve terminal, yet little is known about dephosphorylation processes involved in regulation of synapsin function. Here, we observed a reciprocal relationship in the phosphorylation-dephosphorylation of the established phosphorylation sites on synapsin I. We demonstrate that, in vitro, phosphorylation sites 1, 2, and 3 of synapsin I (P-site 1 phosphorylated by cAMP-dependent protein kinase; P-sites 2 and 3 phosphorylated by Ca(2+)-calmodulin-dependent protein kinase II) were excellent substrates for protein phosphatase 2A, whereas P-sites 4, 5, and 6 (phosphorylated by mitogen-activated protein kinase) were efficiently dephosphorylated only by Ca(2+)-calmodulin-dependent protein phosphatase 2B-calcineurin. In isolated nerve terminals, rapid changes in synapsin I phosphorylation were observed after Ca(2+) entry, namely, a Ca(2+)-dependent phosphorylation of P-sites 1, 2, and 3 and a Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6. Inhibition of calcineurin activity by cyclosporin A resulted in a complete block of Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6 and correlated with a prominent increase in ionomycin-evoked glutamate release. These two opposing, rapid, Ca(2+)-dependent processes may play a crucial role in the modulation of synaptic vesicle trafficking within the presynaptic terminal.

4-Aminopyridine↗
PubMed2001

Mechanisms for increased levels of phosphorylation of elongation factor-2 during hibernation in ground squirrels.

Previously, eEF-2 phosphorylation has been identified as a reversible mechanism involved in the inhibition of the elongation phase of translation. In this study, an increased level of phosphorylation of eukaryotic elongation factor-2 (eEF-2) was observed in the brains and livers of hibernating ground squirrels. In brain and liver from hibernators, eEF-2 kinase activity was increased relative to that of active animals. The activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF-2, was also decreased in brain and liver from hibernators. This was associated with an increase in the level of inhibitor 2 of PP2A (I(2)(PP2A)), although there was an increase in the level of the catalytic subunit of PP2A (PP2A/C) in hibernating brains and livers. These results indicate that eEF-2 phosphorylation represents a specific and previously uncharacterized mechanism for inhibition of the elongation phase of protein synthesis during hibernation. Increased levels of eEF-2 phosphorylation in hibernators appear to be a component of the regulated shutdown of cellular functions that permits hibernating animals to tolerate severe reductions in cerebral blood flow and oxygen delivery capacity.

Animals↗
PubMed2001

Regulation of cyclin-dependent kinase 5 and casein kinase 1 by metabotropic glutamate receptors.

Cyclin-dependent kinase 5 (Cdk5) is a multifunctional neuronal protein kinase that is required for neurite outgrowth and cortical lamination and that plays an important role in dopaminergic signaling in the neostriatum through phosphorylation of Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa). Casein kinase 1 (CK1) has been implicated in a variety of cellular functions such as DNA repair, circadian rhythm, and intracellular trafficking. In the neostriatum, CK1 has been found to phosphorylate Ser-137 of DARPP-32. However, first messengers for the regulation of Cdk5 or CK1 have remained unknown. Here we report that both Cdk5 and CK1 are regulated by metabotropic glutamate receptors (mGluRs) in neostriatal neurons. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist for group I mGluRs, increased Cdk5 and CK1 activities in neostriatal slices, leading to the enhanced phosphorylation of Thr-75 and Ser-137 of DARPP-32, respectively. The effect of DHPG on Thr-75, but not on Ser-137, was blocked by a Cdk5-specific inhibitor, butyrolactone. In contrast, the effects of DHPG on both Thr-75 and Ser-137 were blocked by CK1-7 and IC261, specific inhibitors of CK1, suggesting that activation of Cdk5 by mGluRs requires CK1 activity. In support of this possibility, the DHPG-induced increase in Cdk5 activity, measured in extracts of neostriatal slices, was abolished by CK1-7 and IC261. Treatment of acutely dissociated neurons with DHPG enhanced voltage-dependent Ca(2+) currents. This enhancement was eliminated by either butyrolactone or CK1-7 and was absent in DARPP-32 knockout mice. Together these results indicate that a CK1-Cdk5-DARPP-32 cascade may be involved in the regulation by mGluR agonists of Ca(2+) channels.

Animals↗
PubMed2001

Effects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5.

Cocaine enhances dopamine-mediated neurotransmission by blocking dopamine re-uptake at axon terminals. Most dopamine-containing nerve terminals innervate medium spiny neurons in the striatum of the brain. Cocaine addiction is thought to stem, in part, from neural adaptations that act to maintain equilibrium by countering the effects of repeated drug administration. Chronic exposure to cocaine upregulates several transcription factors that alter gene expression and which could mediate such compensatory neural and behavioural changes. One such transcription factor is DeltaFosB, a protein that persists in striatum long after the end of cocaine exposure. Here we identify cyclin-dependent kinase 5 (Cdk5) as a downstream target gene of DeltaFosB by use of DNA array analysis of striatal material from inducible transgenic mice. Overexpression of DeltaFosB, or chronic cocaine administration, raised levels of Cdk5 messenger RNA, protein, and activity in the striatum. Moreover, injection of Cdk5 inhibitors into the striatum potentiated behavioural effects of repeated cocaine administration. Our results suggest that changes in Cdk5 levels mediated by DeltaFosB, and resulting alterations in signalling involving D1 dopamine receptors, contribute to adaptive changes in the brain related to cocaine addiction.

Animals↗
PubMed2001

Protein phosphatase 1 regulation by inhibitors and targeting subunits.

Regulation of protein phosphatase 1 (PP1) by protein inhibitors and targeting subunits has been previously studied through the use of recombinant protein expressed in Escherichia coli. This preparation is limited by several key differences in its properties compared with native PP1. In the present study, we have analyzed recombinant PP1 expressed in Sf9 insect cells using baculovirus. Sf9 PP1 exhibited properties identical to those of native PP1, with respect to regulation by metals, inhibitor proteins, and targeting subunits, and failure to dephosphorylate a phosphotyrosine-containing substrate or phospho-DARPP-32 (Dopamine and cAMP-regulated phosphoprotein, M(r) 32,000). Mutations at Y272 in the beta12/beta13 loop resulted in a loss of activity and reduced the sensitivity to thiophospho-DARPP-32 and inhibitor-2. Mutations of Y272 also increased the relative activity toward a phosphotyrosine-containing substrate or phospho-DARPP-32. Mutation of acidic groove residues caused no change in sensitivity to thiophospho-DARPP-32 or inhibitor-2, but one mutant (E252A:D253A:E256R) exhibited an increased K(m) for phosphorylase a. Several PP1/PP2A chimeras were prepared in which C-terminal sequences of PP2A were substituted into PP1. Replacement of residues 274-330 of PP1 with the corresponding region of PP2A resulted in a large loss of sensitivity to thiophospho-DARPP-32 and inhibitor-2, and also resulted in a loss of interaction with the targeting subunits, spinophilin and PP1 nuclear targeting subunit (PNUTS). More limited alterations in residues in beta12, beta13, and beta14 strands highlighted a key role for M290 and C291 in the interaction of PP1 with thiophospho-DARPP-32, but not inhibitor-2.

Amino Acid Sequence↗
PubMed2001

Phosphorylation of protein phosphatase inhibitor-1 by Cdk5.

Protein phosphatase inhibitor-1 is a prototypical mediator of cross-talk between protein kinases and protein phosphatases. Activation of cAMP-dependent protein kinase results in phosphorylation of inhibitor-1 at Thr-35, converting it into a potent inhibitor of protein phosphatase-1. Here we report that inhibitor-1 is phosphorylated in vitro at Ser-67 by the proline-directed kinases, Cdk1, Cdk5, and mitogen-activated protein kinase. By using phosphorylation state-specific antibodies and selective protein kinase inhibitors, Cdk5 was found to be the only kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal brain tissue. In vitro and in vivo studies indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by protein phosphatases-2A and -2B. The state of phosphorylation of inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by glutamate-dependent regulation of N-methyl-d-aspartic acid-type channels. Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor of protein phosphatase-1. However, inhibitor-1 phosphorylated at Ser-67 was a less efficient substrate for cAMP-dependent protein kinase. These results demonstrate regulation of a Cdk5-dependent phosphorylation site in inhibitor-1 and suggest a role for this site in modulating the amplitude of signal transduction events that involve cAMP-dependent protein kinase activation.

Animals↗
PubMed2001

TARPP, a novel protein that accompanies TCR gene rearrangement and thymocyte education.

Studies on thymic T cell development have usually concentrated on cell surface molecules. However, intracellular proteins expressed only in thymocytes have never been described. Here we report the discovery of a novel thymocyte-specific protein, named TARPP, which represents a high molecular mass ( approximately 100 kDa) variant of the previously identified protein ARPP-21 ( approximately 21 kDa). TARPP is a cytosolic protein that is expressed at high levels in immature thymocytes. It appears concomitant with the commitment to T cell lineage, and its expression is switched off as a consequence of TCR engagement during positive selection. Such an expression pattern, correlating with the rearrangement of the TCR genes and thymocyte education, suggests a role for TARPP during this important phase of thymocyte development.

Aging↗
PubMed2001

Decreased levels of ARPP-19 and PKA in brains of Down syndrome and Alzheimer's disease.

ARPP-19 (cAMP-regulated phosphoprotein of Mr = 19,000) is a substrate for cAMP-dependent protein kinase (PKA). ARPP-19 is found in all brain regions but the function of ARPP-19 is not fully elucidated yet. We detected a downregulated sequence with 100% homology with ARPP-19 in temporal cortex of patients with Down syndrome (DS) as compared to controls, but not in Alzheimer's disease (AD) using differential displaypolymerase chain reaction (DD-PCR). We subsequently determined protein levels of ARPP-19 in temporal cortex and cerebellum by immunoblotting and observed significant reduction of ARPP-19 in DS (temporal cortex) and AD (cerebellum). We also observed decreased activities of PKA in DS (temporal cortex and cerebellum) and AD (temporal cortex). These findings suggest that decreased ARPP-19 along with decreased activities of PKA is involved in pathomechanisms of both neurodegenerative disorders. Furthermore, these findings provide first evidence for an impaired mechanism of cAMP-related signal transduction and phosphorylation in both dementing disorders.

Alzheimer Disease↗
PubMed2001

Crystal structure of the atypical protein kinase domain of a TRP channel with phosphotransferase activity.

Transient receptor potential (TRP) channels modulate calcium levels in eukaryotic cells in response to external signals. A novel transient receptor potential channel has the ability to phosphorylate itself and other proteins on serine and threonine residues. The catalytic domain of this channel kinase has no detectable sequence similarity to classical eukaryotic protein kinases and is essential for channel function. The structure of the kinase domain, reported here, reveals unexpected similarity to eukaryotic protein kinases in the catalytic core as well as to metabolic enzymes with ATP-grasp domains. The inclusion of the channel kinase catalytic domain within the eukaryotic protein kinase superfamily indicates a significantly wider distribution for this group of signaling proteins than suggested previously by sequence comparisons alone.

Amino Acid Motifs↗